Amplification and suppression of noise in B. subtilis developmental gene circuits

描述（由申请人提供）：“噪声”——细胞成分的随机波动——是单细胞水平上生命的一个基本方面。噪音在生物系统中扮演着双重角色：一方面，它允许细胞根据外部条件做出有偏见的随机决定。例如，通过对压力的孢子反应，单个细胞对未来的环境进行了概率性的“赌注”。另一方面，噪音会干扰依赖于精确基因调控的发育过程。细胞内噪声最近在简单的合成电路中被检测和量化。在这里，我们建议在自然遗传回路中直接分析噪声，这些遗传回路做出概率细胞命运决定并经历精确的发育过程。枯草芽孢杆菌提供了一个独特的机会来做到这一点：它使用特征良好的遗传电路来概率地启动能力和产孢的分化程序。在产孢过程中，它也经历了一个紧密协调、抑制噪音的发育过程。本研究的目的是了解枯草芽孢杆菌基因回路如何放大噪声以概率调节能力事件和孢子形成，以及它们如何抑制噪声以在孢子形成过程中产生有序的事件序列。我们将应用定量延时荧光显微镜技术来观察单细胞中的基因电路动力学，并“重新布线”电路来测试特定的预测。整个方法将由潜在基因回路的数学模型驱动。我们将具体解决三个问题：(1)在能力调节的情况下，我们将测试噪声驱动的兴奋性产生概率性和短暂性能力事件的假设。(2)在产孢启动的情况下，我们将检验嵌套的正反馈回路与噪声一起在产孢启动决策中产生时间变异性的假设。(3)在孢子形成的发育过程中，我们将确定噪音如何在野生型细胞中被抑制，但如何决定部分渗透（PP）突变体（部分细胞成功完成孢子形成，而另一些细胞死亡）的命运。在该模型系统中确定的回路级策略可能在经历分化和发育的更复杂的生物体中起作用。与公共卫生相关：传染病的传播取决于其他遗传程序的概率激活，如细菌的能力、产孢、抗生素的持久性和病毒的潜伏期。这一建议将解决单个细胞随机进入这些交替状态的机制。此外，该提案将研究导致突变部分外显率（仅发生在一些受影响的个体中）的机制。在人类疾病中也发现了部分外显率。

DESCRIPTION (provided by applicant): "Noise" - random fluctuation of cellular components - is a fundamental aspect of life at the single cell level. Noise plays a dual role in biological systems: On the one hand, it allows cells to make random decisions biased by external conditions. For instance, by sporulating in response to stress individual cells make probabilistic 'bets' about their future environment. On the other hand, noise can interfere with developmental processes that depend on precise genetic regulation. Intracellular noise has recently been detected and quantified in simple synthetic circuits. Here we propose to analyze noise directly within natural genetic circuits that make probabilistic cell-fate decisions and undergo precise developmental processes. Bacillus subtilis presents a unique opportunity to do so: It uses well-characterized genetic circuits to probabilistically initiate the differentiation programs of competence and sporulation. It also undergoes a tightly-coordinated, noise-suppressing developmental process during sporulation. The goal of this research is to understand how B. subtilis gene circuits amplify noise to probabilistically regulate competence events and sporulation initiation, and how they suppress noise to generate an ordered sequence of events during sporulation. We will apply quantitative time-lapse fluorescent microscopy techniques to observe gene circuit dynamics in single cells, and 're-wire' circuits to test specific predictions. The overall approach will be driven by mathematical models of underlying gene circuits. We will specifically address three problems: (1) In the case of competence regulation, we will test the hypothesis that noise- driven excitability generates probabilistic and transient competence episodes. (2) In the case of sporulation initiation, we will test the hypothesis that nested positive feedback loops, together with noise, generate temporal variability in the decision to initiate sporulation. (3) In the developmental process of sporulation, we will determine how noise is suppressed in wild-type cells but determines the fate of partially penetrant (PP) mutants (mutants in which some cells successfully complete sporulation, while others die). The circuit-level strategies identified in this model system are likely to operate in more complex organisms that undergo differentiation and development. Relevance to Public Health: The spread of infectious diseases depends on probabilistic activation of alternative genetic programs, such as competence, sporulation, and antibiotic persistence in bacteria, and latency in viruses. This proposal will address the mechanism by which individual cells randomly enter these alternate states. In addition, the proposal will investigate the mechanisms leading to the partial penetrance (occurring only in some affected individuals) of mutations. Partial penetrance is also found in human diseases.